TUBERCULOSIS VACCINE

Abstract: The invention relates to a secreted antigens from mycobacteria capable of evoking early (within 4 days) immunological responses from T-helper cells in the form of gamma-interferon release in memory immune animals after rechallenge infection with mycobacteria of the tuberculosis complex. The antigens are present in short term filtrates (ST-CF) from cultured mycobacteria belonging to the tuberculosis complex. One of these antigens, a polypeptide with an apparent molecular weight of 6 kDa, has been identified, and the DNA encoding the polypeptide has been cloned and sequenced. The antigens of the invention are believed useful especially in vaccines, but also in diagnostic compositions. Also disclosed are nucleic acid fragments encoding the antigens as well as methods of immunizing animals/humans and methods of diagnosing tuberculosis.

Description: TUBERCULOSIS VACCINE The present invention relates to a novel vaccine for immuni¬ zing an animal, including a human being, against tuberculo¬ sis. BACKGROUND Human tuberculosis caused by Mycobacterium tuberculosis is a severe global health problem responsible for approximately 3 million deaths annually (NIH report) . The worldwide incidence of new tuberculosis cases has been progressively falling for the last decade but the recent years has markedly changed this trend due to the advent of AIDS and the appearance of multidrug resistant strains of M. tuberculosis (Rieder) . The only vaccine presently available is BCG, a vaccine which efficacy remains a matter of controversy. BCG generally induces a high level of acquired resistance in animal models of tuberculosis (Smith) , but several human trials in develop¬ ing countries have failed to demonstrate significant protec¬ tion (Fine) . This makes the development of a new and improved vaccine against tuberculosis an urgent matter which has been given a very high priority by the WHO (WHO BULL) . Many attempts to define protective mycobacterial substances have been made, and from 1950 to 1970 several investigators reported an increased resistance after experimental vaccination. However, the demonstration of a specific long-term protective immune response with the potency of BCG has not yet been achieved by administration of soluble proteins or cell wall fragments. Immunity to M. tuberculosis is characterized by three basic features; i) Living bacilli efficiently induces a protective immune response in contrast to killed preparations (Orme) ; ii) Specifically sensitized T lymphocytes mediate this pro¬ tection (Mackaness, Orme) ; iii) The most important mediator molecule seems to be interferon gamma (INF-7) (Rook, Flesh) . Proteins secreted by M. tuberculosis when grown in culture have been demonstrated to function as stimulators of specific cellular immune responses in mice, and it has been suggested that possible antigens useful in new vaccines against tuber- culosis should be sought among such proteins. However, no immune dominant antigen has been isolated or identified. DESCRIPTION OF THE INVENTION It is an object of the present invention to provide a vaccine for immunizing an animal, including a human being against tuberculosis caused by mycobacteria belonging to the tubercu¬ losis-complex. It is demonstrated herein that secreted antigens administered together with an appropriate adjuvant induces specific long- lived Th-1 cells capable of protecting against a subsequent challenge with virulent M. tuberculosis. Importantly, it has surprisingly been found that a vaccine based on soluble polypeptides has the same protective potency as live BCG, especially polypeptides as described below. Consequently, an aspect of the invention is a vaccine for immunizing an animal, including a human being, against tuber¬ culosis caused by mycobacteria belonging to the tuberculosis- complex, comprising as the effective component at least one at least partially purified polypeptide, which is released from metabolizing mycobacteria and present in short-term filtrates from such mycobacteria grown as shaken cultures for 7 days, and has a molecular weight in the range from about 3 to about 16 kDa or in the range from about 20 to about 40 kDa as determined by analysis by SDS-PAGE and silver staining, and induces a release of IFN-7 from reactivated memory T- lymphocytes withdrawn from a C57B1/6J mouse within 4 days after the mouse has been rechallenge infected with myco¬ bacteria belonging to the tuberculosis complex, the induction performed by the addition of the polypeptide to a suspension comprising about 200.000 reactivated memory T-cells per ml, the addition of the polypeptide resulting in a concentration of 1 μg polypeptide per ml suspension, and the release of IFN-7 being assessable by determina- tion of IFN-7 in supernatant harvested 2 days after the addition of the polypeptide to the suspension, or an analogue and/or subsequence of the polypeptide, said analogue and/or subsequence being immunologically equivalent to the polypeptide with respect to the ability of evoking a protective immune response against tuberculosis or with respect to the ability to elicit a delayed type hypersensi- tivity reaction, said polypeptide optionally being coupled to a pharmaceuti¬ cally acceptable carrier or vehicle. The tuberculosis-complex has its usual meaning, i . e . the complex of mycobacteria causing tuberculosis which are Myco- bacterium tuberculosis, Mycobacterium bovis, and Mycobacteri - um africanum. In the present context the term "metabolizing mycobacteria" means live mycobacteria that are multiplying logarithmically and releasing polypeptides into the culture medium. By the term "polypeptide" is herein meant both short peptides with a length of at least two amino acid residues and at most 10 amino acid residues, oligopeptides (11-100 amino acid residues) , and longer peptides (the usual interpretation of "polypeptide", i . e. more than 100 amino acid residues in length) as well as proteins (the functional entity comprising at least one peptide, oligopeptide, or polypeptide which may be chemically modified by being glycosylated, by being lipi- dated, or by comprising prosthetic groups) . The definition of polypeptides also comprises native forms of peptides/proteins in mycobacteria as well as recombinant proteins or peptides in any type of expression vectors transforming any kind of host, and also chemically synthesized peptides. By the terms "analogue" and "subsequence" when used in con¬ nection with polypeptides is meant any polypeptide having the same immunological characteristics as the polypeptides of the invention described above with respect to the ability to confer increased resistance to infections with bacteria belonging to the tuberculosis complex. Thus, included is also a polypeptide from different sources, such as other bacteria or even from eukaryotic cells. The terms "analogue" and "subsequence" with regard to a polypeptide of the invention are also used in the present context to indicate a protein or polypeptide of a similar amino acid composition or sequence as the characteristic amino acid sequence shown in SEQ ID NO: 2, allowing for minor variations which do not have an adverse effect on the ligand binding properties and/or biological function and/or immuno- genicity, or which may give interesting and useful novel binding properties or biological functions and immunogenici- ties etc. The analogous polypeptide or protein may be derived from other microorganisms, cells, or animals and the analogue may also be derived through the use of recombinant DNA tech¬ niques as described below. Furthermore, in the present context the term "immunologically equivalent" means that the analogue or subsequence of the polypeptide is functionally equivalent to the polypeptide with respect to the ability of evoking a protective immune response against tuberculosis and/or eliciting a diagnostically significant immune response (e.g. a Dth reac¬ tion) . The term "protective immune response" has its usual meaning, i.e. that the immune response evoked by the polypeptide in question protects the person immunized from contracting tuberculosis, or that the immune response evoked by the polypeptide at least confers a substantially increased resis¬ tance to infections with mycobacteria belonging to the tuber¬ culosis complex. The ability of the polypeptide to evoke a protective immune response may be assessed by measuring in an experimental animal, e.g. a mouse or a guinea pig, the reduction in myco- bacterial counts from the spleen, lung or other organ homoge- nates isolated from the experimental animal which have received a challenge infection with a virulent strain of Mycobacteriυm tuberculosis after previously having been immunized with the polypeptide, as compared to the mycobacte- rial counts in a control group of experimental animals infected with the same virulent strain of Mycobacterium tuberculosis, which experimental animals have not previously been immunized against tuberculosis. The comparison of the mycobacterial counts may also be carried out with mycobacte- rial counts from a group of experimental animals receiving a challenge infection with the same virulent strain after having been immunized with Mycobacterium bovis BCG. The mycobacterial counts in homogenates from the experimental animals immunized with a polypeptide according to the present invention must at the most be 5 times the counts in the mice or guinea pigs immunized with Mycobacterium bovis BCG, such as at the most 3 times the counts, and preferably at the most 2 times the counts. "Immunologically equivalent" may also mean that the analogue or subsequence is functionally equivalent with the polypeptide with respect to eliciting a delayed type hyper- sensitivity (Dth) reaction to an extent of at least 45% of the Dth reaction elicited by the polypeptide under the same conditions, such as at least 65%, and preferably 85%, measured as the diameter of the Dth reaction. The inventors of the present invention have realised that the polypeptides as defined above are of great importance in evoking a protective immune response against tuberculosis because they induce a release of IFN-7 from reactivated T- lymphocytes from an animal or a human being shortly after the animal or human being have been reinfected. This has been confirmed by measuring the IFN-7 release induced from reactivated T lymphocytes withdrawn from a C57B1/6J mouse within 4 days after the mouse have been rechallenge infected with mycobacteria belonging to the tuberculosis-complex. This is due to the fact that when an immune host mounts a protective immune response, the specific T-cells responsible for the early recognition of the infected macrophage, stimulates a powerful bactericidal activity through their production of IFN-7 (Rook, G.A.W. 1990., Flesch, I. et al. 1987). It is contemplated that because the polypeptides stimulate the T lymphocyte immune response shortly after the onset of the infection they are important in the control of the myco¬ bacteria causing the infection before the mycobacteria have succeeded in multiplying up to the number of bacteria that would have resulted in fulminant infection. Hence, the polypeptides described herein as components in the vaccine are also in their own right an important part of the invention as are the nucleotide fragments encoding the polypeptides of the invention. The polypeptides of interest are within the two main molecu- lar ranges as defined above, but may be within about 4 to about 14 kDa, such as about 6 to about 10 kDa. In a preferred embodiment the polypeptide has a molecular weight in the range of about 5 kDa to about 6 kDa, in the range of about 6 kDa to about 7 kDa or in the range of about 7 kDa to about 8 kDa. The polypeptides may also lie within another molecular inter¬ val which is about 22 to about 38 kDa, or about 20 to about 32 kDa, such as about 22 kDa to about 30 kDa, or even about 24 kDa to about 28 kDa, some other polypeptides may be in the interval of about 35 to about 40 kDa, such as about 36 to about 39 kDa, or even about 36 to about 38 kDa. In a more preferred embodiment the molecular weight may be within about 25 kDa to about 27 kDa or about 37 to about 38 kDa. As described in the examples, three clones of E. coli expres- sing low molecular weight mycobacterial polypeptides from ST- CF have been produced and isolated; all of these proteins are suspected of being involved in the early T-cell responses described above. Thus, according to the invention the polypeptides - produced by the lysogenic E. coli strain designated AA226 which has been deposited 28 June 1993 with the collection of Deutsche Sa mlung von Mikroorganismen und Zellkulturen GmbH (DSM) under the accession number DSM 8377, and/or produced by the lysogenic E. coli strain designated AA227 which has been deposited 28 June 1993 with the collection of Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) under the accession number DSM 8378, and/or produced by the lysogenic E. coli strain designated AA242 which has been deposited 28 June 1993 with the collection of Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) under the accession number DSM 8379 all depo- sitions in accordance with the provisions of the Budapest Treaty, are regarded as interesting aspects of the invention. It has been demonstrated that a low molecular weight protein expressed by the strain AA227 exhibits the immunological profile described above, i.e. has the ability to induce INF-7 release from reactivated memory T-lymphocytes withdrawn from C57B1/6J mice 4 days after rechallenge infection with myco¬ bacteria; this protein is thus an especially preferred aspect of the invention. The protein expressed by this strain binds specifically to a monoclonal antibody designated HYB76-8. Thus, an interesting aspect of the invention is also a polypeptide reacting in a western blot assay with a monoclonal antibody, HYB76-8, said antibody being produced by the hybridoma cell line designated HYB 76-8 0,5/br C8 0,25/br B3 which has been deposited 30 June 1993 with the collection of Deutsche Sammlung von Mikro¬ organismen und Zellkulturen GmbH (DSM) under the accession number DSM ACC2134 in accordance with the provisions of the Budapest Treaty. However, it has been shown by the inventors that the above described immunological profile is not exhibited by any protein expressed by the strain AA226, although this strain expresses another low molecular weight antigen from ST-CF. Therefore, it seems that not all low molecular antigens in ST-CF are directly responsible for or involved in the above- discussed immunological properties of ST-CF, whereas the HYB76-8 reactive antigen must be regarded as one major candi¬ date for the major immunogenic component in a tuberculosis vaccine comprised of single antigens. As discussed below, the mycobacterial antigen expressed by AA226 might possibly have an effect as an "adjuvant" in ST-CF, i.e. the protein is not responsible for the elicitation of the immune response, but has an effect which facilitates the elicitation of efficient immune responses. In a preferred embodiment of the invention the amino acid sequence of the polypeptide comprises an amino acid sequence homologous to the amino acid shown in SEQ ID NO: 2 (cf. also Fig. 10) in the N-terminal part of the sequence or homologous to the amino acid sequence of a analogue and/or subsequence of the amino acid sequence of SEQ ID NO: 2. The term "homologous" is used here to illustrate the degree of identity between the amino acid sequence of a given polypeptide and the amino acid sequence shown in SEQ ID NO: 2 The amino acid sequence to be compared with the amino acid sequence shown in SEQ ID NO: 2 may be deduced from a DNA sequence, e.g. obtained by hybridization as defined below, or may be obtained by conventional amino acid sequencing methods. The degree of homology is preferably determined on the amino acid sequence of a mature polypeptide, i . e. without taking any leader sequence into consideration. It is pre¬ ferred that the degree of homology is at least 80%, such as at least 90%, preferably at least 95% or even 98% with the amino acid sequence shown in SEQ ID NO: 2. Each of the polypeptides may be characterized by specific amino acid and nucleic acid sequences. It will be understood that such sequences include analogues and variants produced by recombinant methods wherein such nucleic acid and polypeptide sequences have been modified by substitution, insertion, addition and/or deletion of one or more nucleoti- des in said nucleic acid sequences to cause the substitution, insertion, addition or deletion of one or more amino acid residues in the recombinant polypeptide. When the term DNA is used in the following, it should be understood that for the number of purposes where DNA can be substituted with RNA, the term DNA should be read to include RNA embodiments which will be apparent for the man skilled in the art. For the purposes of hybridization, PNA may be used instead of DNA, as PNA has been shown to exhibit a very dynamic hybridization profile (PNA is described in Nielsen P E et al . , 1991, Science 254: 1497-1500) . In order to evoke a protective immune response, a polypeptide must be at least 12 amino acids long, preferably at least 15 amino acids, such as 20 amino acids. The nucleotide sequence encoding the above-defined polypeptide may be a nucleotide which 1) is the DNA sequence shown in SEQ ID NO: 1 (shown in Fig. 10) or an analogue and/or subsequence of said sequence which hybridizes with the DNA sequence shown in SEQ ID NO: 1 (or a DNA fragment complementary thereto) or a specific part there¬ of, preferably under stringent hybridization conditions (as defined in the art that is 5-10°C under the melting point T m , cf. Sambrook et al, 1989, pages 11.45-11.49), and/or 2) encodes a polypeptide, the amino acid sequence of which is at least 80% homologous with the amino acid sequence shown in SEQ ID NO: 2, and/or 3) constitutes an effective subsequence of said DNA sequence. The terms "analogue" or "subsequence" when used in connection with the DNA fragments of the invention are intended to indicate a nucleotide sequence which encodes a polypeptide exhibiting identical or substantially identical immunological properties to a polypeptide encoded by the DNA fragment of the invention shown in SEQ ID NO: 1. It is well known that the same amino acid may be encoded by various codons, the codon usage being related, inter alia, to the preference of the organisms in question expressing the nucleotide sequence. Thus, at least one nucleotide or codon of a DNA fragment of the invention may be exchanged by others which, when expressed, result in a polypeptide identical or substantially identical to the polypeptide encoded by the DNA fragment in question. Therefore, the terms "analogue" or "subsequence" are used in the present context to indicate a DNA fragment or a DNA sequence of a similar nucleotide composition or sequence as the DNA sequence encoding the amino acid sequence constitu¬ ting ESAT6 (also denoted "the 6 kDa antigen" or "the HYB76-8 reactive antigen") described herein, allowing for minor variations which do not have an adverse effect on the ligand binding properties and/or biological function and/or immuno- genicity as compared to ESAT6, or which give interesting and useful novel binding properties or biological functions and immunogenicities etc. of the analogue and/or subsequence. The analogous DNA fragment or DNA sequence may be derived from a bacterium, an animal, or a human or may be partially or completely of synthetic origin as described above. The ana¬ logue and/or subsequence may also be derived through the use of recombinant DNA techniques. Furthermore, the terms "analogue" and "subsequence" are intended to allow for variations in the sequence such as substitution, insertion (including introns) , addition, dele¬ tion and rearrangement of one or more nucleotides, which variations do not have any substantial effect on the poly¬ peptide encoded by a DNA fragment or a subsequence thereof. The term "substitution" is intended to mean the replacement of one or more nucleotides in the full nucleotide sequence with one or more different nucleotides, "addition" is under¬ stood to mean the addition of one or more nucleotides at either end of the full nucleotide sequence, "insertion" is intended to mean the introduction of one or more nucleotides within the full nucleotide sequence, "deletion" is intended to indicate that one or more nucleotides have been deleted from the full nucleotide sequence whether at either end of the sequence or at any suitable point within it, and "re- arrangement" is intended to mean that two or more nucleotide residues have been exchanged with each other. A nucleotide subsequence as discussed above refers to an "effective subsequence" which means that it encodes a peptide which is immunologically functional with respect to the ability of evoking a protective immune response against tuberculosis or of eliciting a Dth reaction. The subsequence may be the result of a truncation at either end of the DNA sequence and/or of the removal of one or more nucleotides or nucleotide sequences within DNA sequence. The polypeptide is, as described above, released from the metabolizing mycobacteria, and may therefore be a polypeptide which is translated from a gene in the genome of the mycobac¬ teria. However, the polypeptide released may also be a degra¬ dation product of a larger polypeptide which is catabolized or disintegrated within the mycobacteria whereupon only the products of the catabolization or disintegration are released from the mycobacteria. Therefore, the nucleotide sequence encoding the polypeptide may be part of a larger nucleotide sequence encoding the larger polypeptide present within the living bacteria only. It is in this connection understood that shaken cultures grown for 7 days as described above only represent an insignificantly number of lysed mycobacteria which of course will not secrete any polypeptides but result in release of all (also purely intracellular) polypeptides into the filtrate. A vaccine according to the invention is preferably one which is capable of evoking a substantial and specific acquired immune resistance in a mouse or guinea pig against tuberculo¬ sis caused by mycobacteria belonging to the tuberculosis- complex, which acquired immune resistance corresponds to at least 20% of the protective immune resistance elicited by Mycobacterium bovis BCG, as assessed by the observed reduc¬ tion in mycobacterial counts from spleen, lung or other organ homogenates isolated from the mouse or guinea pig receiving a challenge infection with a virulent strain of M. tuberculo- sis, as described above. The preferred acquired immune resistance corresponds to at least 50% of the protective immune response elicited by M. bovis BCG, such as at least 60%, or even more preferred to at least 80% of the protective immune response elicited by M. Jovis BCG, such as at least 90%. When compared to the immune response elicited by M. bovis BCG it is possible in a preferred aspect of the present invention that the vaccine confers to the person vaccinated an acquired immune resistance corresponding to at least 100% of the protective immune response elicited by M. bovis BCG, such as at least 110%. Preparation of vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all incorpor¬ ated herein by reference. Typically, such vaccines are pre¬ pared as injectables either as liquid solutions or suspen¬ sions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients which are pharma¬ ceutically acceptable and compatible with the active ingredi¬ ent. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines. The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscu¬ larly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%. Oral formulations include such normally employed excipients as, for example, pharma¬ ceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredi¬ ent, preferably 25-70%. The proteins may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium,, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeuti- cally effective and immunogenic. The quantity to be adminis¬ tered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of protection desired. Suit¬ able dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 μg to 1000 μg, such as in the range from about 1 μg to 300 μg, and especially in the range from about 10 μg to 50 μg. Suitable regimes for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations. The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologi- cally acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the age of the person to be vaccinated and, to a lesser degree, the size of the person to be vaccinated. Some of the polypeptides of the vaccine are sufficiently immunogenic in a vaccine, but for some of the others the immune response will be enhanced if the vaccine further comprises an adjuvant substance. Various methods of achieving adjuvant effect for the vaccine include use of agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as 0.25 percent solution, aggrega¬ tion of the protein in the vaccine by heat treatment with temperatures ranging between 70° to 101°C for 30 second to 2 minute periods respectively. Aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopoly- saccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono- oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed. According to the invention DDA (dimethyldi- octadecylammonium bromide) is an interesting candidate for an adjuvant, but also Freund's complete and incomplete adjuvants as well as QuilA and RIBI are interesting possibilities. Other possibilities involve the use of immune modulating substances such as lymphokines (e.g. IFN-7, IL-2 and IL-12) or synthetic IFN-7 inducers such as poly I:C in combination with the above-mentioned adjuvants. As discussed in example 3, it is contemplated that such mixtures of antigen and adjuvant will lead to superior vaccine formulations. In many instances, it will be necessary to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at least about three vaccinations. The vaccinations will normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years, usually three years, will be desirable to maintain the desired levels of protective immunity. The course of the immunization may be followed by in vitro proliferation assays of PBL (peripheral blood lymphocytes) co-cultured with ESAT6 or ST-CF, and especially by measuring the levels of IFN-7 released form the primed lymphocytes. The assays may be performed using conventional labels, such as radionuclides, enzymes, fluorescers, and the like. These techniques are well known and may be found in a wide variety of patents, such as U.S. Patent Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of these types of assays. As described above a measurement of the effect of the polypeptides in the vaccine may be to assess the IFN-7 released from memory T-lymphocytes. The stronger immune response the more IFN-7 will be released, accordingly, a vaccine according to the invention comprises a polypeptide capable of releasing from the memory T-lymphocytes at least 1500 pg/ l, such as 2000 pg/ml, preferably 3000 pg/ml IFN-7. Due to genetic variation different individuals may react with immune responses of varying strength to the same polypeptide. Therefore, the vaccine according to the invention may com¬ prise several different polypeptides in order to increase the immune response. The vaccine may comprise two or more polypeptides, where all of the polypeptides are as defined above, or some but not all of the peptides may be derived from a bacterium belonging to the Af. tuberculosis complex. In the latter example the polypeptides not necessarily fulfil¬ ling the criteria set forth above for polypeptides may either act due to their own immunogenicity or merely act as adju¬ vants. Examples of such interesting polypeptides are MPB64, MPT64, the ST-3 reactive polypeptide, the PV-2 reactive polypeptide, and MPB59, but any other substance which can be isolated from mycobacteria are possible candidates. The vaccine may comprise 3-20 different polypeptides, such as 3-10 different polypeptides. One reason for admixing the polypeptides of the invention with an adjuvant is to effectively activate a cellular immune response. However, this effect can also be achieved in other ways, for instance by expressing the effective antigen in a vaccine in a non-pathogenic microorganism. A well-known example of such a microorganism is Mycobacterium bovis BCG. Therefore, another important aspect of the present invention is an improvement of the BCG vaccine presently available, which is a vaccine for immunizing an animal, including a human being, against tuberculosis caused by mycobacteria belonging to the tuberculosis-complex, comprising as the effective component a microorganism, wherein one or more copies of a DNA sequence encoding a polypeptide as defined above or an analogue and/or subsequence thereof has been incorporated into the genome of the microorganism in a manner allowing the microorganism to express and secrete the polypeptide. In the present context the term "genome" refers to the chro¬ mosome of the microorganisms as well as extrachromosomally DNA or RNA, such as plasmids. The assessment of the capability of the vaccine with respect to evoking a protective immune response being as defined above, as well as the assessment of the effect of the vaccine as compared to conventional BCG vaccine. The microorganism in the vaccine may be a bacterium such as bacteria selected from the group consisting of the genera Mycobacterium, Salmonella, Pseudomonas and Eschericia. In a preferred embodiment the microorganism is AfycoJbacteriu-iϊ Jbovis BCG, and in a more preferred embodiment it is Mycobac¬ terium bovis BCG strain: Danish 1331, which is the Mycobacte¬ rium bovis strain Copenhagen from the Copenhagen BCG Labora¬ tory, Statens Seruminstitut, Denmark. The incorporation of one or more copies of a DNA sequence encoding the polypeptide according to the invention in a mycobacterium from a Af. bovis BCG strain will enhance the immunogenic effect of the BCG strain especially with respect to the long-term immune response as described above. The incorporation of more than one copy of the DNA sequence is contemplated to enhance the immune response even more, consequently an aspect of the invention is a vaccine wherein at least 2 copies of a DNA sequence encoding a polypeptide is incorporated in the genome of the microorganism, such as at least 5 copies. The copies of DNA sequences may either be identical encoding identical polypeptides or be variants of the same DNA sequence encoding identical or homologues of a polypeptide, or in another embodiment be different DNA sequences encoding different polypeptides where at least one of the polypeptides is according to the present invention. The DNA sequence used in such an embodiment of the invention may be a DNA sequence which 1) is identical to the DNA sequence shown in SEQ ID NO: 1 or an analogue and/or subsequence which hybridizes with the DNA sequence shown in SEQ ID NO: 1 (or a DNA sequence complementary thereto) or a specific part thereof, pre¬ ferably under stringent hybridization conditions and/or 2) encodes a peptide, the amino acid sequence of which is at least 80% homologous with the amino acid sequence shown in SEQ ID NO: 2, and/or 3) constitutes an effective subsequence of said DNA sequence, the definitions of analogue and subsequence being as defined above. The DNA sequence is according to the invention preferably one that encodes a peptide the amino acid sequence of which is at least 90% homologous with the amino acid sequence shown in SEQ ID NO: 2 or with a subsequence thereof, or encodes the peptide with the amino acid sequence shown in SEQ ID NO: 2, or encodes a subsequence thereof. As discussed above, the elicitation of cell-mediated responses to infection can be obtained by employing an adjuvant or by using live vaccines. However, recent research have revealed a new an exciting possibility, wherein a DNA fragment cloned in a vector which is non-replicative in eukaryotic cells is introduced into an animal (including a human being) by e.g. intramuscular injection or percutaneous administration. The DNA is taken up by e.g. muscle cells and the gene of interest is expressed by a promoter which is functioning in eukaryotes, e.g. a viral promoter, and the gene product thereafter stimulates the immune system. These newly discovered methods are reviewed in Ulmer et al., 1993, which hereby is included by reference. Therefore, also a part of the invention is a vaccine compri¬ sing a nucleic acid fragment according to the invention, the vaccine effecting in vivo expression of antigens by an ani- mal, including a human being, to whom the vaccine has been administered, the amount of expressed antigens being effec¬ tive to confer substantially increased resistance to infec¬ tions with mycobacteria of the tuberculosis complex in an animal, including a human being. Also, methods of immunizing animals against tuberculosis by administering the vaccine to the animals are parts of the invention. The efficacy of such a "DNA vaccine" can possibly be enhanced by administering the gene encoding the expression product together with a DNA fragment encoding a polypeptide which has the capability of modulating an immune response. For instance, a gene encoding lymphokine precursors or lympho- kines (e.g. IFN-7, IL-2, or IL-12) could be administered together with the gene encoding the immunogenic protein, either by administering two separate DNA fragments or by administering both DNA fragments included in the same vector. In both immunodiagnostics and vaccine preparation, it is often possible and practical to prepare antigens from seg- ments of a known immunogenic protein or polypeptide. Certain epitopic regions may be used to produce responses similar to those produced by the entire antigenic polypeptide. Potential antigenic or immunogenic regions may be identified by any of a number of approaches, e.g., Jameson-Wolf or Kyte-Doolittle antigenicity analyses or Hopp and Woods (1981) hydrophobicity analysis (see, e.g., Jameson and Wolf, 1988; Kyte and Doo- little, 1982; or U.S. Patent No. 4,554,101). Hydrophobicity analysis assigns average hydrophilicity values to each amino acid residue from these values average hydrophilicities can be calculated and regions of greatest hydrophilicity deter¬ mined. Using one or more of these methods, regions of pre¬ dicted antigenicity may be derived from the amino acid sequence assigned to the polypeptides of the invention. Therefore, yet another aspect of the present invention is the polypeptide as defined above, especially one which comprises an epitope for a T-helper cell. Examples of such polypeptides are those produced by the deposited E. coli strains described above. Furthermore, the invention relates to a nucleotide fragment comprising a nucleotide sequence encoding a polypeptide as defined above, such as a nucleotide sequence encoding any one of the polypeptides produced by the deposited E. coli strains described above, especially the nucleotide fragment which comprises the DNA sequence of SEQ ID NO: 1. Yet another aspect of the invention is a composition for diagnosing tuberculosis, comprising a polypeptide as defined above, especially the polypeptides produced by the deposited E. coli strains described above, such as the polypeptide encoded by a nucleotide fragment which comprises the DNA sequence of SEQ ID NO: 1 or a part thereof, or the composi¬ tion comprising a nucleotide sequence as defined above. Methods of determining the presence of mycobacterial anti- bodies or components of mycobacteria in samples or in animals are also parts of the invention, as a method of determining the presence of antibodies directed against mycobacteria belonging to the tuberculosis complex in an animal, including a human being, or in a sample, comprising administering a polypeptide of the invention to the animal or incubating the sample with the polypeptide of the invention, and detecting the presence of bound antibody resulting from the administra¬ tion or incubation. Likewise, a method of determining the presence of a mycobacterial antigen in an animal, including a human being, or in a sample, comprising administering an antibody of the invention to the animal or incubating the sample with the antibody, and detecting the presence of bound antigen resulting from the administration or incubation, forms part of the invention. Finally a method of determining the presence of mycobacterial nucleic acids in an animal, including a human being, or in a sample, comprising adminis¬ tering a nucleic acid fragment of the invention to the animal or incubating the sample with the nucleic acid fragment of the invention or a nucleic acid fragment complementary there- to, and detecting the presence of hybridized nucleic acids resulting from the incubation, is also included in the inven- tion. Such a method of diagnosing tuberculosis might involve the use of a composition comprising at least a part of a nucleotide sequence as defined above and detecting the pre¬ sence of nucleotide sequences in a sample from the animal or human being to be tested which hybridize with to the nucleotide fragment (or a complementary fragment) by the use of PCR technique. Preferred immunoassays are contemplated as including various types of enzyme linked immunoassays (ELISAS) , immunoblot techniques, and the like, known in the art. However, it readily appreciated that utility is not limited to such assays, and useful embodiments include RIAs and other non-en¬ zyme linked antibody binding assays or procedures. It is contemplated that several assays for the presence of mycobacteria or for TB may be developed using any of the polypeptides of the invention, the corresponding nucleic acid fragments encoding the protein, functionally similar proteins and their epitopes, or by detection of other appropriate nucleic acids. Reactive epitopes representing portions of the polypeptide sequences could be utilized in an analogous manner. Finally, diagnostic kits for the diagnosis of on-going or previous TB infection forms part of the invention. The diag¬ nostic kits of the invention comprises an antibody, a nucleic acid, or a polypeptide according to the invention in combina¬ tion with a means for detecting the interaction with the relevant substance reacting with these substances of the invention; the choice of these detection means is discussed below with reference to DNA fragments, but it will be under- stood that the same considerations apply for polypeptides and monoclonal antibodies of the invention. In both the diagnostic methods, compositions, and kits the antibodies, nucleic acids or polypeptides according to the invention may optionally be coupled to solid or semi-solid carriers, as is well-known in the art. In clinical diagnostic embodiments, nucleic acid segments of the present invention may be used in combination with an appropriate means, such as a label, to determine hybridiza¬ tion with DNA of a pathogenic organism. Typical methods of detection might utilize, for example, radioactive species, enzyme-active or other marker ligands such as avidin/biotin, which are detectable directly or indirectly. In preferred diagnostic embodiments, one will likely desire to employ an enzyme tag such as alkaline phosphatase or peroxidase rather than radioactive or other reagents that may have undesirable environmental effects. Enzyme.tags, for example, often uti¬ lize colorimetric indicator substrates that are readily detectable spectrophotometrically, many in the visible wave¬ length range. Luminescent substrates could also be used for increased sensitivity. Hybridizable DNA segments may include any of a number of segments of the disclosed DNA. For example, relatively short segments including 12 or so base pairs may be employed, or, more preferably when probes are desired, longer segments including 20, 30 or 40 base pairs, depending on the particu¬ lar applications desired. Shorter segments are preferred as primers in such applications as PCR, while some of the longer segments are generally preferable for blot hybridizations. It should be pointed out, however, that while sequences dis¬ closed for the DNA segments of the present invention are defined by SEQ ID NO: l a certain amount of variation or base substitution would be expected, e.g., as may be found in mutants or strain variants, but which do not significantly affect hybridization characteristics. Such variations, inclu¬ ding base modifications occurring naturally or otherwise, are, as mentioned above intended to be included within the scope of the present invention. As mentioned, in certain aspects, the DNA sequence informa¬ tion provided by the invention allows for the preparation of relatively short DNA (or RNA or PNA) sequences having the ability to specifically hybridize to mycobacterial gene sequences. In these aspects, nucleic acid probes of an appro¬ priate length are prepared based on a consideration of the sequence, e.g., SEQ ID NO: 1. The ability of such nucleic acid probes to specifically hybridize to the mycobacterial gene sequences lend them particular utility in a variety of embodiments. Most importantly, the probes can be used in a variety of diagnostic assays for detecting the presence of pathogenic organisms in a given sample. However, either uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructs. To provide certain of the advantages in accordance with the invention, the preferred nucleic acid sequence employed for hybridization studies or assays includes sequences that are complementary to at least a 10 to 40, or so, nucleotide stretch of the selected sequence, such as that shown in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 13. A size of at least 10 nucleotides in length helps to ensure that the fragment will be of sufficient length to form a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 10 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. Thus, one will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 20 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesi¬ zing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR tech- nology of U.S. Patent 4,603,102, or by introducing selected sequences into recombinant vectors for recombinant produc¬ tion. As can be seen from example 6, the polypeptides of the inven¬ tion are also capable of eliciting a Dth response in the form of a skin reaction in guinea pigs. The polypeptides of the invention may thus be useful as agents in a diagnostic skin test. Therefore, the invention also relates to a method of diagno¬ sing tuberculosis caused by Mycobacterium tuberculosis, Mycobacterium africanum or AycoJbacteriuπi Jbovis in an animal, including a human being, comprising intradermally injecting, in the animal, a pharmaceutical composition containing a polypeptide as defined above or an analogue and/or subsequence thereof which is immunologically equivalent to the peptide, a positive skin response at the location of injection being indicative of the animal having tuberculosis, and a negative skin response at the location of injection being indicative of the animal not having tuberculosis. A further aspect of the invention is a method for immunising a mammal, including a human being, against tuberculosis caused by mycobacteria belonging to the tuberculosis-complex, wherein a vaccine as defined above is administered to the mammal, the vaccine may be administered intravenously, intra- peritoneally, intracutaneously or intramuscularly, in doses well-known to the person skilled in the art (cf. the dis¬ cussion of administration of the vaccines of the invention above) . Another aspect is a monoclonal antibody, which is substan¬ tially specifically reacting with a polypeptide as defined above in an immune assay, such as a Western blot or an ELISA test. In a preferred embodiment the monoclonal antibody is expressed by the deposited cell-line which has been deposited by the applicant 30 June 1993 with the collection of Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) under the accession number DSM ACC2134, or a specifically binding fragment of said antibody. Yet a another aspect is a nucleotide sequence encoding the antibody described above, such as the nucleotide sequence which is contained in the deposited cell-line described above. Another aspect is a replicable vector which expresses a polypeptide as defined above. The vector may be any vector which may conveniently be subjected to recombinant DNA pro- cedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i . e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication; examples of such a vector are a plasmid, phage, cosmid, mini-chromo¬ some or virus. Alternatively, the vector may be one which, when introduced in a host cell, is integrated in the host cell genome and replicated together with the chromosome(s) into which it has been integrated. Expression vectors may be constructed to include any of the DNA segments disclosed herein. Such DNA might encode an antigenic protein specific for virulent strains of mycobac¬ teria or even hybridization probes for detecting mycobacteria nucleic acids in samples. Longer or shorter DNA segments could be used, depending on the antigenic protein desired. Epitopic regions of the proteins expressed or encoded by the disclosed DNA could be included as relatively short segments of DNA. A wide variety of expression vectors is possible including, for example, DNA segments encoding reporter gene products useful for identification of heterologous gene products and/or resistance genes such as antibiotic resis¬ tance genes which may be useful in identifying transformed cells. Recombinant vectors such as those described are particularly preferred for transforming bacterial host cells. Accordingly, a method is disclosed for preparing transformed bacterial host cells that includes generally the steps of selecting a suitable bacterial host cell, preparing a vector containing a desired DNA segment and transforming the selected bacterial host cell. Several types of bacterial host cells may be employed, including E. coli , B. subtilis, as well as rapid- growing mycobacteria such as M. smegmatis or even BCG. Also other prokaryotic and eukaryotic host cells may be employed. Transformed cells may be selected using various techniques, including screening by differential hybridization, identifi¬ cation of fused reporter gene products, resistance markers, anti-antigen antibodies and the like. After identification of an appropriate clone, it may be selected and cultivated under conditions appropriate to the circumstances, as for example, conditions favouring expression or, when DNA is desired, replication conditions. The present invention therefore further relates to a cell harbouring at least one replicable expression vector as defined above. In principle, this cell may be of any type of cell, i . e. a prokaryotic cell such as a bacterium, e.g. E. coli or a Mycobacterium tuberculosis, or Mycobacterium bovis, or Mycobacterium africanum, a unicellular eukaryotic organism, a fungus or yeast, or a cell derived from a multicellular organism, e.g. an animal or a plant. It is especially in cases where glycosylation is desired that a mammalian cell is used, although glycosylation of proteins is a rare event in prokaryotes. The cell may preferably by a cell which is selected from the group consisting of the lysogenic E. coli strains AA226, AA227 and AA242 which have been deposited 28 June 1993 with the collection of Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) under the accession numbers DSM 8377, DSM 8378 and DSM 8379, respectively, in accordance with the provisions of the Budapest Treaty, or a M. tuberculosis bovis BCG cell. A further aspect of the invention is a method for producing a polypeptide as defined above comprising inserting a DNA fragment as defined above into a vector which is able to replicate in a host cell, introducing the resulting recombi¬ nant vector into the host cell, culturing the host cell in an appropriate culture medium under appropriate conditions for expressing the polypeptide, and recovering the polypeptide from the host cell or culture medium and optionally subjec¬ ting the recovered polypeptide to post-translational modifi¬ cations, or by isolating the polypeptide from short-term culture filtrate as defined herein. The medium used to grow the cells may be any conventional medium suitable for the purpose. A suitable vector may be any of the vectors described above, and an appropriate host cell may be any of the cell types listed above. The methods employed to construct the vector and effect introduction thereof into the host cell may be any methods known for such purposes within the field of recombinant DNA. In the follow¬ ing a more detailed description of the possibilities will be given: In general, of course, prokaryotes are preferred for the initial cloning of nucleic sequences of the invention and constructing the vectors useful in the invention. For example, in addition to the particular strains mentioned in the more specific disclosure below, one may mention by way of example, strains such as E. coli K12 strain 294 (ATCC No. 31446), E. coli B, and E. coli X 1776 (ATCC No. 31537). These examples are, of course, intended to be illustrative rather than limiting. Prokaryotes are also preferred for expression. The aforementioned strains, as well as E. coli W3110 (F-, lamb¬ da-, prototrophic, ATCC No. 273325), bacilli such as Bacillus subtilis, or other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may be used. Especially interesting are rapid-growing mycobacteria, e.g. Af. smegmatis, as these bacteria have a high degree of resemblance with mycobacteria of the tubercu¬ losis complex and therefore stand a good chance of reducing the need of performing posttranslational modifications of the expression product. In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (see, e.g., Bolivar et al., 1977, Gene 2: 95). The pBR322 plasmid contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to con¬ tain, promoters which can be used by the microorganism for expression. Those promoters most commonly used in recombinant DNA con¬ struction include the B-lactamase (penicillinase) and lactose promoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel et al., 1979) and a tryptophan (trp) promoter system (Goeddel et al., 1979; EPO Appl. Publ. No. 0036776). While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally with plasmid vectors (Siebwenlist et al., 1980). Certain genes from proka¬ ryotes may be expressed efficiently in E. coli from their own promoter sequences, precluding the need for addition of another promoter by artificial means. In addition to prokaryotes, eukaryotic microbes, such as yeast cultures may also be used. Saccharomyces cerevisiase, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979; Tschemper et al., 1980). This plasmid already contains the trpl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan for example ATCC No. 44076 or PEP4-1 (Jones, 1977) . The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective envi- ronment for detecting transformation by growth in the absence of tryptophan. Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzman et al., 1980) or other glycolytic enzymes (Hess et al., 1968; Holland et al., 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phospho- fructokinase, glucose-6-phosphate isomerase, 3-phosphoglyce- rate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expres¬ sion vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination. Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promo¬ ter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeast- compatible promoter, origin of replication and termination sequences is suitable. In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts. In prin- ciple, any such cell culture is workable, whether from verte¬ brate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate in culture (tissue culture) has become a routine procedure in recent years (Tissue Culture, 1973) . Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COS-7 293 and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences. For use in mammalian cells, the control functions on the expression vectors are often provided by viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40) . The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of repli- cation (Fiers et al., 1978). Smaller or larger SV40 fragments may also be used, provided there is included the approximate¬ ly 250 bp sequence extending from the Hindlll site toward the Bgll site located in the viral origin of replication. Fur¬ ther, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems. An origin of replication may be provided either by construc¬ tion of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) or may be provided by the host cell chromosomal replication mechanism. If the vector is inte¬ grated into the host cell chromosome, the latter is often sufficient. In the light of the above discussion the methods for recombi- nantly producing the polypeptide of the invention are also a part of the invention, as are the vectors carrying and/or being capable of replicating the nucleic acids according to the invention in a host cell or a cell-line. According to the invention the expression vector can be e.g. a plasmid, a cosmid, a minichromosome, or a phage. Especially interesting are vectors which are integrated in the host cell/cell line genome after introduction in the host. After the recombinant preparation of the polypeptide accor¬ ding to the invention, the isolation of the polypeptide may for instance be carried out by affinity chromatography (or other conventional biochemical procedures based on chromato¬ graphy) , using a monoclonal antibody which substantially specifically binds the polypeptide according to the inven¬ tion. Another possibility is to employ the simultaneous electroelution technique described by Andersen et al. in J. Immunol. Methods 161: 29-39. According to the invention the post-translational modifica- tions involves lipidation, glycosylation, cleavage, or elon¬ gation of the polypeptide. The DNA sequence to be modified may be of cDNA or genomic origin as discussed above, but may also be of synthetic origin. Furthermore, the DNA sequence may be of mixed cDNA and genomic, mixed cDNA and synthetic or genomic and syn¬ thetic origin as discussed above. The DNA sequence may have been modified, e.g. by site-directed mutagenesis, to result in the desired DNA fragment encoding the desired polypeptide. The following discussion focused on modifications of DNA encoding the polypeptide should be understood to encompass also such possibilities, as well as the possibility of buil¬ ding up the DNA by ligation of two or more DNA fragments to obtain the desired DNA fragment, and combinations of the above-mentioned principles. The DNA sequence may be modified using any suitable technique which results in the production of a DNA fragment encoding a polypeptide of the invention. The modification of the DNA sequence encoding the amino acid sequence of the polypeptide of the invention should be one which does not impair the immunological function of the resulting polypeptide. A preferred method of preparing variants of the antigens disclosed herein is site-directed mutagenesis. This technique is useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, derived from the antigen sequences, through specific mutage¬ nesis of the underlying DNA. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing conside¬ rations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucle- otide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleo¬ tides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered. In general, the technique of site-specific mutagenesis is well known in the art as exemplified by publications (Adelman et al., 1983). As will be appreciated, the technique typical- ly employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage (Messing et al., 1981). These phage are readily commer- cially available and their use is generally well known to those skilled in the art. In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector which includes within its sequence a DNA sequence which encodes the polypeptides of the invention. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example by the method of Crea et al. (1978) . This primer is then annealed with the single- stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement. The preparation of sequence variants of the selected nucleic acid fragments of the invention using site-directed mutagene- sis is provided as a means of producing potentially useful species of the genes and is not meant to be limiting as there are other ways in which sequence variants of the nucleotide fragments of the invention may be obtained. For example, recombinant vectors encoding the desired genes may be treated with mutagenic agents to obtain sequence variants (see, e.g., a method described by Eichenlaub, 1979) for the mutagenesis of plasmid DNA using hydroxylamine. Analogues/subsequences of the disclosed nucleic acid frag¬ ments which also form part of the invention are nucleic acid fragments which are fused to at least one other nucleic acid fragment which encodes a protein enhancing the immunogenicity of the fused protein relative to a protein without the encoded fusion partner. Such encoded proteins may e.g. be T- cell epitopes or other immunogenic epitopes enhancing the immunogenicity of the target gene product, e.g. lymphokines such as INF-7, IL-2 and IL-12. Other nucleic acid fragments to form part of a nucleic acid fragment of the invention encoding a fusion polypeptide are those encoding polypeptides which facilitate expression and/or purification of the fused peptide, e.g. bacterial fimbrial proteins, e.g. the pilus components pilin and papA; protein A; the ZZ-peptide; the maltose binding protein; gluthatione S-transferase; β-galactosidase; or poly- histidine. The polypeptide of the invention may alternatively be pro¬ duced by the well-known methods of solid or liquid phase peptide synthesis utilizing the successive coupling of the individual amino acids of the polypeptide sequence or coup¬ ling of individual amino acids forming fragments of the polypeptide sequence so as to result in the desired polypeptide. A yet further aspect of the invention is a method for produ¬ cing a vaccine according as defined above, comprising producing or isolating a polypeptide as defined above, and solubilizing or dispersing the polypeptide in a medium for a vaccine, and optionally adding other M. tuberculosis antigens and/or an adjuvant substance, or cultivating a cell from a microorganism comprising at least one nucleotide sequence as described above, and transferring the cell to a medium for a vaccine, and optionally adding an adjuvant substance. LEGENDS TO FIGURES Fig. 1: Course of infection with M. tuberculosis in naive and memory immune mice. C57B1/6J mice were infected with 2.5 x 10 5 viable units of M. tuberculosis and the growth of the organisms in the spleen was investigated for a period of 25 days. The count of the CFU indicated represent the means of 4-5 mice. Fig. 2: In vivo IFN-7 production during tuberculosis infec¬ tion. Memory immune or naive mice were infected with 2.5 x 10 5 colony forming units of Af. tuberculosis i.v. and the level of IFN-7 was monitored in the spleen or serum of animals during the course of infection. Fig. 3: In vitro response of spleen lymphocytes from infected mice. Memory immune or naive mice were sacrificed at different time points during the course of infection, and spleen lymphocytes were stimulated in vitro with ST-CF or killed bacilli. Cell culture supernatants were tested for the presence of IFN-7. Fig. 4: Short-term culture-filtrate fractions. ST-CF was divided into 14 fractions by the multi-elution technique and the fractions were analyzed by SDS-PAGE and Silver-staining. Lane F: ST-CF Lane 1-15: fractions 1-15. Fig. 5: T-cell reactivation during a secondary infection. IFN-7 release by spleen lymphocytes isolated either directly from memory immune mice or four days after the mice had received a secondary infection. The lymphocytes were stimu¬ lated in vitro with ST-CF fractions and the supernatants harvested for quantification of IFN-7. The migration of molecular mass markers (as shown in Fig. 4) are indicated at the bottom. Fig. 6: Precise mapping of IFN-7 release in response to single secreted antigens. A panel of narrow fractions within the stimulatory regions 4- 14 and 26-34 enabled the precise mapping of proteins capable of inducing IFN-7 in microcultures containing lymphocytes from memory immune mice at day 4 of rechallenge. On the left hand side: IFN-7 release by single secreted antigens. On the right hand side: The localization of and IFN-7 induc- tion by defined secreted antigens of M. tuberculosis. ST-3, 76-8 and PV-2 are the designation of three mAbs which defines secreted antigens of molecular mass 5-8 kDa. Fig. 7: Biochemical purification of HYB76-8 reactive antigen (ESAT6) . SDS-PAGE analysis of purified ESAT6 obtained from a three step purification method involving a final gel filtration on a Superdex 75 column. The sample applied to the column was the HYB76-8 reactive antigen containing fraction eluted from the Mono Q column during the 2nd purification step. Lanes 5, 6, and 7 show the presence of the HYB76-8 reactive antigen in the region below the 14.4 kDa molecular weight marker. Fig. 8: Physical map of recombinant lambda phages expressing products reactive with Mabs recognizing low MW components. Cross-hatched bar; lacZ, solid bar; Af. tuberculosis DNA, open bar; lambdagtll DNA (right arm) , open triangles indicate EcoRI cleavage sites originating from the lambdagtll vector. The direction of translation and transcription of the gene products fused to beta-galactosidase is indicated by an arrow. Fig. 9: Western blot analyses demonstrating recombinant expression of low molecular weight components. Lysates of E. coli Y1089 lysogenized with lambda AA226, lambda AA227 or lambda were analyzed in Western blot experi- ments after PAGE (A: 10%, B: 10 to 20% gradient) . Panel A: lanes 1: lambda gtll, lanes 2: lambda AA226, lanes 3: lambda AA227. Panel B: lane 1: lambda gtll, lanes 2 and 3: lambda AA242 and AA230 (identical clones) . The monoclonal antibodies are indicated on top of each panel. L24,c24 is an anti-MPT64 reactive monoclonal antibody. Figs. 10A, 10B, and 10C: Recombinant ESAT6. 10A: Plasmid map of pAA249. The 1.7 kbp EcoRl - BamHl fragment of lambda AA227 subcloned into EcoRI - BamHl sites of pBluescript. Cross-hatched bar; mycobacterial DNA, Arrow; the esatβ gene. 10B: The complete DNA sequence of the mycobacterial DNA of pAA249. The sequence is obtained by the dideoxy sequencing method (Sanger, F. et al. 1977, DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. 74 : 5463) and cycle se¬ quencing using the Dye Terminator system in combination with the automated gel reader, model 373A from Applied Biosystems; the sequence is also shown in SEQ ID NO: 1. ESAT6 is encoded by the DNA sequence from the ATG start codon at position 13 - 15 to the TAG stop codon at position 298 - 300. 10C: The deduced amino acid sequence of ESAT6 (also shown in SEQ ID NO: 2) in conventional three letter code. The * indi¬ cate amino acids which could be aligned to the sequence obtained by N-terminal sequencing of biochemically purified native material. Fig. 11: ST-CF fractions for investigation of T cell response patterns in genetically heterogeneous animals. ST-CF were separated in SDS-PAGE and silver stained. MW markers are indicated to the left and the cut-offs used in the preparation of fractions 1-10 to the right. These frac- tions were used for the experiments indicated in Figs. 12 and 13. Figs. 12A and 12B: Guinea pig responses to ST-CF fractions. Lymphocyte stimulation results with spleen lymphocytes (Fig. 12A) and peripheral blood lymphocytes (Fig. 12B) . The cells were not stimulated (C) or stimulated with 1 μg of fractions 1-10 (F1-F10) or with PPD and ST-CF. The stimulations are means from groups of 8-10 guinea pigs sensitized as indicated in the figure insert. Fig. 13: T cell responses in different strains of inbreed mice. Mouse T cells were stimulated in vitro with the panel of ST- CF fractions and IFN-7 release to the culture supernatants monitored. Fig. 14: Human IFN-7 response to ST-CF fractions. Human PBL were stimulated in vitro with ST-CF fractions and cell culture supernatants investigated for the presence of IFN-7. Fig. 15: The skin test inducing capacity of purified, native ESAT6 in aerosol infected guinea pigs. The diameter of skin test reactions was measured 3, 6, 8, and 11 days after exposure of 4 groups of guinea pigs (N=5) to aerosols of M. tuberculosis. PREAMBLE TO EXAMPLES It is an established fact that long-term immunological memory resides after termination of a tuberculous infection (Orme, I.M. 1988., Lefford, M.J. et al. 1974.). This memory immunity efficiently protects the host against a secondary infection with M. tuberculosis later in life. When an immune host mounts a protective immune response, the specific T-cells responsible for the early recognition of the infected macrophage, stimulates a powerful bactericidal activity through their production of IFN-7 (Rook, G.A.W. 1990., Flesch, I. et al. 1987.). Protective antigens which are to be incorporated in a future sub-unit vaccine have in the examples below been sought among the molecular targets of the effector cells responsible for the recall of a protective immune response. This has resulted in the identification of immunodominant antigenic targets for T-cells during the first phase of a protective immune response. Bacteria. M. tuberculosis H37Rv (ATCC 27294) was grown at 37°C on Lδwenstein-Jensen medium or in suspension in modified Sauton medium. BCG Copenhagen was obtained as a freeze dried vaccine and were rehydrated with diluted sauton followed by a brief sonication to ensure a disperse suspension. Production of short-term culture filtrate .ST-CF) . ST-CF was produced as described previously (Andersen et al., 1991b). Briefly M. tuberculosis (4 x 10 6 CFU/ml) were incubated in Sauton medium and grown on an orbital shaker for 7 days. The bacteria were removed by filtration and the culture superna¬ tants were passed through sterile filters (0.2 μ ) and con¬ centrated on an Amicon YM 3 membrane (Amicon, Danvers, Mass.) . Fractionation of ST-CF by the multi-elution technique. ST-CF (5 mg) was separated in 10-20% SDS-PAGE overnight (11 cm vide centerwell, 0.75 mm gel). After the termination of the elec- trophoretic run the gel was trimmed for excess gel, and pre- equilibrated in 3 changes of 2 mM phosphate buffer for 40 min. The multi-elution was performed as described previously (Andersen and Heron, 1993b) . Briefly, gels were transferred to the Multi-Eluter™ (KEM-EN-TECH) and electroeluted (40 V) into 2 mM phosphate buffer for 20 min. The polypeptide frac¬ tions were aspirated and adjusted to isotonia with concen¬ trated PBS. All fractions were stabilized with 0.5% mice serum and were kept frozen at -80°C until use. Lymphocyte cultures. Lymphocytes were obtained by preparing single-cell suspensions from spleens as described in Andersen et al., 1991a. Briefly, ST-CF or antigenic fractions were added to microcultures containing 2 x 10 5 lymphocyte in a volume of 200 μl Rpmi 1640 supplemented with 5 x 10 5 M 2- mercaptoethanol, penicillin, streptomycin, 1 mM glutamine and 0.5% (vol/vol) fresh mouse serum. ST-CF was used in the concentration 4 μg/ml while ST-CF fractions were used in 1 μg/ml. Cellular proliferation was investigated by pulsing the cul¬ tures (1 μCi [ 3 H] thymidine/well) after 48 h of incubation, further incubating the plates for 22 hours and finally har¬ vesting and processing the plates for liquid scintillation counting (Lkb, Beta counter) . Culture supernatants were harvested from parallel cultures after 48 hours incubation and used for lymphokine analyses. Lymphokine analyses. The amount of INF-7 present in culture supernatants and in homogenised organs was quantified by an IFN-7 ELISA kit (Holland Biotechnology, Leiden, the Nether¬ lands) . Values below 10 pg were considered negative. EXAMPLE 1 Isolation of T-cell stimulating low molecular weight ST-CF antigens A group of efficiently protected mice was generated by infec- ting 8-12 weeks old female C57B1/6J mice bred at Statens Seruminstitut, Copenhagen, Denmark, with 2.5 x 10 3 M. tuber¬ culosis i.v. After 30 days of infection the mice were sub¬ jected to 60 days of antibiotic treatment with isoniazid and were then left for 200-240 days to ensure the establishment of resting long-term memory immunity. The mice were then reinfected with 2.5 x 10 5 JVf. tuberculosis i.v. and the course of infection was compared with that of a corresponding naive group of mice (Fig. 1) . As seen in Fig. 1, M. tuberculosis grow rapidly in the spleens of naive mice whereas the infection is controlled . within the first few days in memory immune mice. This finding emphasizes that early immunological events occurring during the first days determines the outcome of infection. Gamma interferon (IFN-7) is a lymphokine which is involved directly in protective immunity against Af. tuberculosis (Rook G. A. W. , 1990, Flesch I. and Kaufmann S., 1987). To monitor the onset of a protective immune response, the content of IFN-7 in spleen homogenates (4% w/v in PBS) and in serum samples was investigated during the course of infection (Fig. 2) . Memory immune mice were found to respond immediately (<24 h) by a marked production of IFN-7 detectable both in spleen and in serum. Naive mice, in contrast, had a 14 days delay before any significant production was evident, a period during which infection rapidly progressed. Immune mice were characterized by an accelerated release of IFN-7 and to determine the molecular targets of this immunological response, spleen lymphocytes were obtained from animals at different time points during the course of infection. The lymphocytes were stimulated in vitro with either bacteria, killed with glutaraldehyde and washed with PBS or short-term culture-filtrate (ST-CF) which is a complex mixture of pro¬ teins secreted by M. tuberculosis during growth (Andersen, P. et al. 1991.) (Fig. 3). The memory immune mice were found to be characterized by an accelerated generation of IFN-7 pro¬ ducing T-cells responding to ST-CF whereas killed bacteria in contrast were found to elicit only a marginal response at a very late stage of infection. To map the molecular targets of protective T-cells among the multiple secreted proteins present in ST-CF a screening of ST-CF was performed using the multi-elution technique (Andersen and Heron, 1993b) . This technique divides complex protein mixtures separated in SDS-PAGE into narrow fractions in a physiological buffer (Fig. 4) . These fractions were used to stimulate spleen lymphocytes in vitro and the release of IFN-7 was monitored (Fig. 5) . The response of long-term memory immune mice (the mice were left for 200-240 days to ensure immunological rest) was compared to the response generated after 4 days of rechallenge infection. This com- parison enable the mapping of targets for memory effector T- cells triggered to release IFN-7 during the first phase of a protective immune response. Using this approach it was demon¬ strated that the targets for these protective T-cells were secreted proteins or fragments of proteins of apparent mole- cular mass 6-10 and 26-34 kDa (Fig. 5) . To precisely map single molecules within the stimulatory regions the induction of IFN-7 by a panel of narrow overlap¬ ping fractions was investigated. This enabled the identifica¬ tion of a 6-8 kDa protein fraction with exceedingly stimula- tory capacity (5100-5400 pg IFN-7 units/ml) (Fig. 6) . The 6- kDa protein band yielding the highest release of IFN-7 (5390 pg/ml) was recognized by the mAb HYB76-8, whereas the adja¬ cent protein bands were recognized by the Abs ST-3 and PV-2. The mAb HYB76-8 which therefore defines the major target for protective T-cells was used to identify the antigen during a 3 step column chromatography: 1. Hydrophobic interaction chromatography. 3M ammonium sulphate in phosphate buffer was added to ST-CF (lane 1, Fig. 7) (1-10 mg/ml) so as to obtain a final concen¬ tration of 1M ammonium sulphate. The precipitated proteins were removed by centrifugation, and the supernatant applied to a Phenyl Sepharose CL-4B (Pharmacia) column (Pharmacia Fine Chemicals, Uppsala, Sweden) . Western blot analysis at this point shows that the HYB76-8 reactive antigen was pri¬ marily present in the supernatant. A linear gradient of decreasing concentration of ammonium sulphate (a linear gradient 1M - 0M ammonium sulphate, in 50 mM phosphate buffer (pH 8.5) was run. The HYB76-8 reactive antigen was eluted at concentrations of 100 mM - 0 mM ammonium sulphate. 2. Anion exchange chromatography. Fractions containing HYB76-8 reactive antigen were collected and submitted to buffer exchange. The buffer used was a 50 mM TRIS/HC1, pH 8.5. The sample was run on a Mono Q (Pharmacia) using a linear gradient of 0 - 0.5 NaCl. The HYB76-8 reactive antigen was eluted at a NaCl concentration of 150-200 mM NaCl. 3. Gel filtration. HYB76-8 reactive antigen fractions were applied directly to a Superdex 75 16/60 column (Pharmacia) , 3 ml per run. The buffer used was PBS, pH 7.4. Half of the HYB76-8 reactive antigen containing fractions collected contain GroES as the only other antigen (lanes 5, 6, and 7 in Fig. 7) . Results obtained from experiments similar to those of example 1 have further pointed to the protein recognized by the mAb HYB76-8 as an important antigen in the early response to reinfection with M. tuberculosis. In experiments with T-cell suspensions from rechallenged mice it was demonstrated that chemically purified HYB76-8 reactive antigen elicited an exceedingly high release of IFN-7 (13933±836 pg/ml at a protein concentration of 20 μg/ml) . ST-CF in comparison did in this experiment induce a release of 7300±208 pg/ml. Recom¬ binant HYB76-8 reactive antigen did in such experiments elicit a somewhat lower release of IFN-7(3500-5000 pg/ml). EXAMPLE 2 Vaccination with a ST-CF containing vaccine The results in example 1 pinpoints ST-CF as target for T- cells involved in protective immunity, a finding which was further confirmed by investigating the protective efficacy of an experimental vaccine based on ST-CF: Female, 8-12 weeks old C57B1/6J mice bred at Statens Serum Institute, Copenhagen, Denmark, were immunized with 5 x 10 4 CFU of BCG s.c in 0.2 ml saline at the base of the tail. This dose was found to induce an optimal protective immune response in our animal model (results not shown) . The experimental vaccines which contained 100 mg ST-CF/dose and applied dimethyldioctadecylammonium bromide (DDA) (250 mg/dose) as adjuvant in 0.2 ml were given S.C. three times with weekly intervals at different sites on the back of the mice to boost a strong cellular immune response to ST-CF. DDA (Eastmann Kodak, USA) was prepared by suspension of the powder in distilled water (2.5 mg ml "1 ) . A fine homogenous dispersion of the powder was obtained by heating the suspen¬ sion to 80°C for 5-10 minutes. After cooling at room tempera- ture the suspension was mixed with equal amount of either PBS or diluted ST-CF. Each injection contained 250 μg of DDA in 0.2 ml. In the first series of protection experiments the mice were left for 12-14 weeks after the first injection and were then challenged by an i.v. injection of 1 x 10 4 viable Af. tubercu ¬ losis. The course of the disease was monitored in the spleens and lungs at different time points during the first 28 days. In the second series of protection experiments the mice were challenged 5-6 weeks after the first injection by an i.p. injection of 1 x 10 6 Af. tuberculosis. After 2-3 weeks of infection the mice were killed and the number of viable bacteria in the spleens of infected mice was determined by plating double serial 10-fold dilutions of organ homogenates on Lδwenstein-Jensen medium. Colonies were counted after 3 to 4 weeks of incubation, and the data were expressed as the log 10 values of the geometric means of counts obtained with six to twelve mice (Table 1) . TABLE 1 Bacterial numbers in organs of vaccinated mice receiving a challenge of virulent M. tuberculosis Experiment 1 Experiment 2 Immunization 8 Spleen Lung Spleen Control 5 .41 3 .61 4 .83 BCG 4 .00 (P=0 .0001) 2 .94 (P=0 .0012) 2 .96 (P=0 .0002 ) ST-CF and PBS 5 .48 3 .70 ND PBS and DDA 5 .18 4 .03 4 .38 ST-CF and DDA 4 .17 (P=0 .0001) 2 .93 (P=0 .0026) 3 .46 (P=0 . 0042 ) * Mice were immunized with BCG or injected three times with the experi ¬ mental vaccines . b Bacterial numbers are expressed as the log 10 values of the geometric means (n = 6 in experiment 1 and n = 12 in experiment 2) . SEM is less than 0 .16 in experiment 1 and less than 0 .32 in experiment 2 . P values have been given for bacterial numbers that are significantly different from the numbers found for unimmunized control animals . These experiments convincingly demonstrated that ST-CF con¬ tains protective antigens which can be used to boost a long- term memory immune response of the same protective efficacy as the one provided by live BCG . EXAMPLE 3 Construction of a vaccine based on selected secreted antigen fractions . The molecular targets for INF-7 producing T cells involved in the first phase of a protective immune response were found within the region 6-10 and 26-34 kDa of ST-CF (Fig. 5) . An experimental vaccine based on these selected antigen frac¬ tions and the adjuvant DDA was therefore constructed and tested in our animal model. In addition this experiment included vaccines based on the two single antigenic targets identified so far;the 31-32 kDa antigen 85 and a recombinant version of the 6 kDa antigen. Vaccinated mice were left for 12-14 weeks after the first injection and were then challenged by an i.v. injection of lxlO 4 viable M. tuberculosis. Mice were killed and bacteria enumerated as in the previous e_-q?eriment (table 2) . TABLE 2. Bacterial numbers in spleens of mice vaccinated with fractions of ST-CF and challenged with Af. tuberculosis. Immunization 8 *" Bacterial numbers 0 -' Control 5.17 BCG 3.51 PBS and DDA 4.75 ST-CF (6-10 kDa) and DDA 4.33 ST-CF (26-34) and DDA 4.02 Ag 85 and DDA 4.85 REC HYB76-8 reactive antigen 4.90 and DDA a) Mice were immunized with BCG or injected three times with experimental vaccines. b) Bacterial numbers are expressed as the log 10 values of the geometric means. SEM are less than 0.30. The experiment demonstrated that both the vaccine based on the antigenic fractions ranging from 6-10 and the vaccine based on the antigenic fractions from 26-34 kDa induced an increased level of acquired resistance, a result which empha¬ sizes the presence of protective antigens within these regions of ST-CF. Neither purified Ag 85 or the 6 kDa antigen, however induced any significant protection in this experiment. The reason for the low efficacy of these vaccines based on the purified products was pursued by investigating the T cell subsets induced by the immunization. T cells were isolated from the vaccinated mice and stimulated in vitro with ST-CF. Cellular proliferation in the cultures were investigated and the release of IFN-7 quantified. This experiment demonstrated that although all the different vaccination protocols induced T cells proliferating in response to ST-CF in vitro exceed¬ ingly low levels of IFN-7(<50 pg/ml) were present in the cultures with T cells from mice immunized with the purified proteins (the 6 kDa and Ag 85) . Cells derived from mice immunized with a mixture of the complex mixture of ST-CF and DDA in contrast, released 1800-2000 pg/ml of IFN-7. This result strongly suggests that whereas a vaccine based on the mixture of proteins contained within ST-CF induces a protective immune response consisting predominantly of Th-1 cells characterized by the release of high levels of IFN-7 and IL-2 and low levels of IL-4 and IL-5 (results not shown) , a similar vaccine based on single purified products are characterized by the induction of a response biased towards non-protective Th-2 cells not capable of producing IFN-7. A possible explanation on this discrepancy may be that mol- ecules with adjuvant properties (i munomodulators) exist among the proteins present in ST-CF. The work has therefore been continued with the purpose of establishing an adjuvant system capable of inducing a powerful IFN-7 response, even with purified products. DDA has been combined with recombi- nant IFN-7 or the synthetic IFN-7 inducer poly-IC. These mixtures have been mixed with ST-CF, mice immunized and the reactivity of the T cells induced has been investigated by stimulating T cell suspensions in vitro with ST-CF (table 3) . TABLE 3 Recall reactivity in Vitro after immunization with ST-CF in different adjuvant combinations. This experiment demonstrated that both of these additions induced an enhanced T cell proliferative response associated with markedly increased levels of IFN-7. This line of research will be continued (eg. by the addition of other recombinant cytokines) or testing of alternative adjuvant systems. The criteria used for determining the feasibility of an adjuvant will be the induction of an effi- cient Th-1 response as judged by: 1) High IFN-7/IL-4 ratio during in vitro recall response. 2) High IFN-7/IL-4 mRNA ratio induced in the regional lymph- nodes. 3) High IgG2a/IgGl ratio in specific immunoglobulin induced by the immunization. 4) High efficacy of the vaccine against a subsequent chal¬ lenge. The purified proteins will subsequently be tested in the optimal adjuvant combination. EXAMPLE 4 Cloning of genes expressing HYB76- 8, PV-2 and ST- 3 binding proteins . It was demonstrated (in example 1) that low molecular weight components (components with an apparent molecular weight less than that of GroES) in short-term culture filtrate reacted with the monoclonal antibodies produced by the three hybridomas ST-3, HYB76-8, and PV-2. In order to identify the antigens binding to these antibodies, the following experi- ments were carried out: The recombinant λgtll Af. tuberculosis DNA library constructed by R. Young (Young, R.A. et al. 1985) and obtained through the World Health Organization IMMTUB programme (WHO.0032.wibr) was screened for phages expressing gene products which would bind the monoclonal antibodies HYB76-8, PV-2 and ST-3. Approximately 1 x 10 5 pfu of the gene library (containing approximately 25% recombinant phages) were plated on Escher- icia coli Y1090 (ΔlacU169, proA + , Δlon, araD139, supF, trpC22: : tnlO [pMC9] ATCC#37197) in soft agar and incubated for 2,5 hours at 42°C. The plates were overlaid with sheets of Isopropyl-/3-D-thioga- lacto pyranoside saturated sheets of nitrocellulose and incubation was continued for 2,5 hours at 37°C. The nitrocellulose was removed and incubated with samples of the monoclonal antibodies in PBS with Tween 20 added to a final concentration of 0.05%. Bound monoclonal antibodies were visualized by horseradish peroxidase-conjugated rabbit anti- mouse immunoglobulins (P260, Dako, Glostrup, DK) and a stai- ning reaction involving 5,5', 3,3' tetramethyl benzidine and H 2 0 2 . Positive plaques were recloned and the phages originating from a single plaque were used to lysogenize E. coli Y1089 (ΔlacU169, proA + , Δlon, araD139, strA, hfll50 [chr::__πlO] [pMC9] ATCC nr. 37196) . The resultant lysogenic strains were used to propagate phage particles for DNA extraction. These lysogenic E. coli strains have been deposited in the German Collection of Microorganisms and Cell Cultures in Brauns¬ chweig, FRG under the DSM-Accession numbers: DSM 8377 = AA226 (expressing ST-3 reactive polypeptide) , DSM 8378 = AA227 (expressing HYB76-8 reactive polypeptide) , DSM 8379 = AA242 (expressing PV-2 reactive polypeptide) . A physical map of the recombinant phages is shown in Fig. 8 and the expression of the recombinant gene products is shown Fig. 9. The HYB76-8 and ST-3 binding proteins are expressed as fusion proteins fused to / 3-galactosidase whereas the PV-2 binding protein appear to be expressed in an unfused version. Sequencing of the nucleotide sequence encodincr the HYB76-8 binding protein. In order to obtain the nucleotide sequence of the gene encoding the HYB76-8 binding protein the 1.7 kbp M. tuberculosis derived EcoRI - BamHl fragment from AA227 was subcloned in pBluescriptSK+ (Stratagene, La Jolla, Ca.) (Fig. 10A) and used to transform E. coli XL-lBlue (Stratagene, La Jolla, Ca.) . The complete DNA sequence obtained by the dideoxy sequencing method (Sanger, F. et al. 1977, 'DNA sequencing with chain terminating inhibitors'. Proc. Natl. Acad. Sci. 74: 5463) and cycle sequencing using the Dye Terminator system in combina¬ tion with the automated gel reader, model 373A from Applied Biosystems, is shown in Fig. 10B. An open reading frame encoding a sequence of 95 amino acid residues was identified from an ATG start codon at position 13 - 15 extending to a TAG stop codon at position 298 - 300. The deduced amino acid sequence is shown in Fig. IOC using conventional three letter code. The * indicate amino acids which could be aligned to the sequence obtained after N- terminal sequencing of biochemically purified native material. Comparison of deduced and observed amino acid composition of ESAT6. (The aminoacids are compared to Leucine which was assigned the value = 1) . observed deduced from DNA sequence No homologous sequences were found by searching the GenEMBL databases using the Sequence Analysis Software Package ver¬ sion 7.1 from the Genetics Computer Group associated with the University of Wisconsin (Devereux, J. et al. 1984) . The HYB76-8 binding protein is therefore believed to be novel. EXAMPLE 5 Immunodominance of the low molecular weight secreted antigens The stimulatory potential of secreted protein fractions was investigated in outbred guinea pigs and human donors. This was done to analyze the possible influence of genotype and experimental design on the relative immunodominance of the 6- 10 kDa secreted antigen fraction. T cell responses in different strains of inbreed mice. Gene- tically different strains of inbreed mice representing different MHC kl 2 haplotypes were infected with Af. tubercu¬ losis for 14 days. Splenic T cells were isolated and stimu- lated with the panel of secreted protein fractions (Fig. 11) . The release of IFN-7 in the cultures were quantified and the response pattern of the different strains compared (Fig. 13) . Five out of six strains of mice demonstrated a predominant recognition of fraction 1 a result which further supported the notion that this fraction is generally immune-dominant during the live infection. Stimulatory capacity of ST-CF fractions in sensitized guinea pigs. Groups of outbred guinea pigs from strains Ssc:AL were sensitized by infection with Af. tuberculosis, BCG i.d., BCG i.v., or immunized with killed AT. tuberculosis in oil or (as a control) with oil alone. Af. tuberculosis H37Rv and AT. bovis BCG Copenhagen were used. When infected with Af. tuberculosis H37Rv guinea pigs were given 2.5 x 10 3 cfu in a volume of 0.1 ml in an ear vein. Infection by the same route (i.v.) with BCG was done with 2.5 x 10 4 cfu. Vaccinations with BCG were done with four intradermal (i.d.) injections on the abdomen of 0.1 ml recon¬ stituted BCG vaccine. BCG Copenhagen contained approximately 4 x 10 6 cfu per ml of the reconstituted preparation. Immuni- zations with killed bacteria were given 4 x 0.1 ml i.d. on the abdomen of a suspension of glutaraldehyde killed bacteria at 0.4 mg (semidry weight) per ml of paraffine oil (Marcol 52 (M52) ) - 3-4 weeks later peripheral blood and spleen lymphocytes were isolated and used for stimulation experiments with a set of 10 fractions of ST-CF (prepared as in: Andersen & Heron, 1993a) . The MW of these fraction appears from Fig. 11. Peripheral blood lymphocytes were isolated from blood drawn by cardiac puncture using EDTA as anticoagulant. Erythrocytes were removed by ficoll density gradient (d = 1.09) centrifugation. Lymphocytes were washed twice, counted and the cell concentration adjusted to 2 x 10 6 cells/ml in RPMI 1640 with supplements including 5% FCS. Spleen lymphocytes were isolated by pressing spleens through a wire mesh. Erythrocytes were lysed by treatment with 0.84% NH 4 C1. The lymphocytes were washed twice and the cell concentration adjusted to 2 x 10 6 cells/ml of RPMI with supplements. 0.1 ml of cells were cultured with 0.1 ml of antigen or mitogen in triplicate for 6 days, the last 22 h in the pre¬ sence of 1 μCi 3 H-thymidine. Cultures were harvested and incorporated 3 H-thymidine was counted in a scintillation counter. Results are calculated as geometric means of tripli¬ cate cultures, and geometric means between guinea pigs within immunization groups are shown in Figs. 12A and 12B. There was no significant stimulation of PBL or SPL from control (M52, oil alone) immunized guinea pigs. For both cell types it is evident that infection with Af. tuberculosis or BCG leads to a highly significant superior sensitivity to fraction 1 than to the other fractions. With the exception of peripheral blood lymphocytes from the BCG i.d. group which showed a peak for fractions 5-7 and 10, there was no note- worthy differences between responses to fraction 2-10 in the infected animals. When immunizing with killed Af. tuberculo¬ sis, peak responses to fraction 1, 7, and 10 were seen for both cell types. It should be noted that this group is the only one in which fraction 1 does not give a significantly higher response than all other fractions, thereby supporting the general conclusion that responses to the low molecular mass secreted antigens is associated with the live infection. These results underline the importance of the <10000 Da region in the response to infection with AT. tuberculosis "complex" mycobacteria. IFN-7 release in human lymphocyte cultures stimulated with ST-CF fraction. Peripheral blood mononuclear cells (PBMC) were obtained from heparinized venous blood from human donors, diluted 1:1 in saline, and separated by sedimentation over Lymphoprep (Nycomed A/S, Oslo, Norway) density gradient centrifugation. Cells were collected, washed twice and cul¬ tured in flat-bottomed microtiter plates (Nunc, Roskilde, Denmark) , at 5 x 10 4 cells per well, in a volume of 200 μg of RPMI 1640 containing 10% human AB serum and ST-CF fractions (1-2 μg/ml.). Supernatants were harvested from the lymphocyte cultures at day 5. The amount of IFN-7 present was quantified by an IFN-7 enzyme-linked immunosorbent assay kit (Holland Biotechnology, Leiden, The Netherlands) . Patients with newly diagnosed active Tb were found to be characterized by a marked production of IFN-7 to a range of ST-CF fractions. This is in contrast to the other donor groups (BCG vaccinated and patients with advanced disease) which demonstrated a rather limited reactivity to secreted protein fractions (Fig. 14) . Secreted proteins of molecular mass 6-10 kDa were found to posses a superior IFN-7 inducing capability in the active Tb patients and elicited a mean release of 35 u/ml (6125 pg/ml) . Conclusions. ST-CF is a mixture of antigens secreted by M. tuberculosis during growth. ST-CF is herein demonstrated to contain protective antigens which can be administered as an experimental vaccine and evoke the same level of specific acquired resistance as live BCG. The immunodominant T-cell antigen in this complex mixture has been identified in three different models. Human patients with newly diagnosed tuberculosis as well as guinea pigs and mice infected with a virulent strain of M. tuberculosis respond powerfully to secreted antigens and the most potent fraction containing proteins ranging from 6-10 kDa. In long-term memory immune mice it is demonstrated that one of the major targets for memory effector T-cells triggered during the first phase of a protective immune response is the same low molecular mass fraction. A protein band within this fraction responsible for the pronounced reactivity has been identified as a 6 kDa protein antigen defined by the mAb HYB76-8. A procedure has been devised for the purification of this protein and the gene encoding the protein has been cloned and sequenced. Both biochemically purified and recombinant HYB76-8 reactive antigen were demonstrated to posses the powerful IFN-7 indu¬ cing capacity. A vaccine based on a chemically purified protein fraction highly enriched in the 6 kDa antigen induced substantial levels of protection whereas vaccines based on purified recombinant HYB76-8 reactive antigen provoked pre¬ dominantly TH-2 responses of low protective efficacy. Experi¬ ments are in progress to optimize the adjuvant used, thereby allowing the monitoring of the protective efficacy of purified proteins too. EXAMPLE 6 ESAT6 as a diagnostic agent in a sJin test. In order to test the possible use of ESAT6 as a diagnostic agent, the following experiment was carried out: Four groups of guinea pigs (n=5) were exposed to aerosols of Af. tuberculosis Erdman at doses giving rise to an average of 5 primary tuberculous lesions per lung. Skin testings were performed after 3, 6, 8, and 11 weeks after inhalation with 1 μg of purified ESAT6. As can be seen from Fig. 15 a positive skin test reaction ( > 5 mm) was observed in all guinea pigs 11 weeks after infection. A clear division in a responding and a non-respon¬ ding group was observed at earlier time points. REFERENCES Andersen, P., D. Askgaard, L. Ljungqvist, J. Bennedsen, I. Heron. 1991a, T-cell proliferative response to antigens secreted by ycoJbacteriuπ. tuberculosis. 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SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Statens Seruminstitut (B) STREET: Artillerivej 5 (C) CITY: Copenhagen (E) COUNTRY: Denmark (F) POSTAL CODE (ZIP) : 2300 S (ii) TITLE OF INVENTION: Novel tuberculosis vaccine (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1753 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 13..300 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAATTCCAAA AC ATG ACA GAG CAG CAG TGG AAT TTC GCG GGT ATC GAG 48 Met Thr Glu Gin Gin Trp Asn Phe Ala Gly lie Glu 1 5 10 GCC GCG GCA AGC GCA ATC CAG GGA AAT GTC ACG TCC ATT CAT TCC CTC 96 Ala Ala Ala Ser Ala lie Gin Gly Asn Val Thr Ser lie His Ser Leu 15 20 25 CTT GAC GAG GGG AAG CAG TCC CTG ACC AAG CTC GCA GCG GCC TGG GGC 144 Leu Asp Glu Gly Lys Gin Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly 30 35 40 GGT AGC GGT TCG GAG GCG TAC CAG GGT GTC CAG CAA AAA TGG GAC GCC 192 Gly Ser Gly Ser Glu Ala Tyr Gin Gly Val Gin Gin Lys Trp Asp Ala 45 50 55 60 ACG GCT ACC GAG CTG AAC AAC GCG CTG CAG AAC CTG GCG CGG ACG ATC 240 Thr Ala Thr Glu Leu Asn Asn Ala Leu Gin Asn Leu Ala Arg Thr lie 65 70 75 AGC GAA GCC GGT CAG GCA ATG GCT TCG ACC GAA GGC AAC GTC ACT GGG 288 Ser Glu Ala Gly Gin Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly 80 85 90 ATG TTC GCA TAGGGCAACG CCGAGTTCGC GTAGAATAGC GAAACACGGG 337 Met Phe Ala 95 ATCGGGCGAG TTCGACCTTC CGTCGGTCTC GCCCTTTCTC GTGTTTATAC GTTTGAGCGC 397 ACTCTGAGAG GTTGTCATGG CGGCCGACTA CGACAAGCTC TTCCGGCCGC ACGAAGGTAT 457 GGAAGCTCCG GACGATATGG CAGCGCACGC GTTCTTCGAC CCCAGTGCTT CGTTTCCGCC 517 GGCGCCCGCA TCGGCAAACC TACCGAAGCC CAACGGCCAG ACTCCGCCCC CGACGTCCGA 577 CGACCTGTCG GAGCGGTTCG TGTCGGCCCC GGCCGCCACC CCCCCACCCC CACCTCCGCC 637 TCCGCCAACT CCGATGCGAT CGCGCAGGAG AGCCGCCCTC GCCGGAACCG GCCGCATCTA 697 AACCACCCAC ACCCCCCATG CCCATCGCCG GACCCGAACC GGCCCCACCC AAACCACCCA 757 CACCCCCCAT GCCCATCGCC GGACCCGAAC CGGCCCCACC CAAACCACCC ACACTCCGAT 817 GCCCATCGCC GGACCTGCAC CCCACCCAAC GAATCCCAGT TGGCGCCCCC CAGACCACCG 877 ACACCACAAA CGCCAACCGG AGCGCCGCAG CAACCGGAAT CACCGGTGCC CCACGTACCC 937 TCGCACGGGC CACATCAACC CCGGTGCACC GCACCAGCAC CGCCCTGGGC AAAGATGCCA 997 ATCGGCGAAC CCCCGCCCGC CCGTCCAGAC CGTCTGCGTC CCCGGCCGAA CCACCGACCC 1057 GGCCTGCCCC CCAACACTCC CGACGTGCGC GCCGGGGTCA CCGCTATCGC ACAGACACCG 1117 AACGAAACGT CGGGAAGGTA GCAACTGGTC CATCCATCCA GGCGCGGCTG CGGGCAGAGG 1177 AAGCATCCGG CGCGCAGCTC GCCCCCGGAA CGGAGCCCTC GCCAGCGCCG TTGGGCCAAC 1237 CGAGATCGTA TCTGGCTCCG CCCACCCGCC CCGCGCCGAC AGAACCTCCC CCCAGCCCCT 1297 CGCCGCAGCG CAACTCCGGT CGGCGTGCCG AGCGACGCGT CCACCCCGAT TTAGCCGCCC 1357 AACATGCCGC GGCGCAACCT GATTCAATTA CGGCCGCAAC CACTGGCGGT CGTCGCCGCA 1417 AGCGTGCAGC GCCGGATCTC GACGGAACAG AAATCCTTAA GCCGGCGCGA AGGGGCCGCA 1477 AGGTGAAGAA GGTGAAGCCC CAGAAACCGA AGGCCACGAA GCCGCCCAAA GTGGTGTCGC 1537 AGCGCGGCTG GCGACATTGG GTGCATGCGT TGACGCGAAT CAACCTGGGC CTGTCACCCG 1597 ACGAGAAGTA CGAGCTGGAC CTGCACGCTC GAGTCCGCCG CAATCCCCGC GGGTCGTATC 1657 AGATCGCCGT CGTCGGTCTC AAAGGTGGGG CTGGCAAAAC CACGCTGACA GCAGCGTTGG 1717 GGTCGACGTT GGCTCAGGTG CGGGCCGACC GGATCC 1753 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 95 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Thr Glu Gin Gin Trp Asn Phe Ala Gly lie Glu Ala Ala Ala Ser 1 5 10 15 Ala lie Gin Gly Asn Val Thr Ser lie His Ser Leu Leu Asp Glu Gly 20 25 30 Lys Gin Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser 35 40 45 Glu Ala Tyr Gin Gly Val Gin Gin Lys Trp Asp Ala Thr Ala Thr Glu 50 55 60 Leu Asn Asn Ala Leu Gin Asn Leu Ala Arg Thr lie Ser Glu Ala Gly 65 70 75 80 Gin Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala 85 90 95 Applicant's or agent's tile 201 International applies tionNi reference number :T/DK 94 / 0 0273 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule I3bis) The indications made below relate to the microorganism referred to in the description on page , line B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | χ{ Name of depositary institution Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) Address of depositary institution (including postal code and country) Maschroder eg lb D-38124 Braunschweig Federal Republic of Germany- Date of deposit Accession Number 28 June 1993 DSM 8377 C. ADDITIONAL INDICATIONS (leave blank ifnol applicable) This information is continued on an additional sheet Q As regards the respective Patent Offices of the respective designated states, the applicant requests that a sample of the deposited microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if tlie indications are not for all designated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the international Bureau litaispcafyihegcnminatureof'heindicationse.g^ 'Acce on Number of Deposit ') For International Bureau use only I i This sheet was received by the international Bureau on: Authorized officer Applicant's or agent's file 31201 International application N reference number :T/DK 94 / 0 0273 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule I3bis) A. The indications made below relate to the microorganism referred to in the description 7 ,. 24-27 on page _______ _________________ , line _______________________ B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet j >j Name of depositary institution Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) Address of depositary institution (including postal code and country) Maschroder Weg lb D-38124 Braunschweig Federal Republic of Germany Date of deposit Accession Number DSM 33-3 28 June 1993 C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet [~ As regards the respective Patent Offices of the respective designated states, the applicant requests that a sample of the deposited microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if tlie indications are not for all designated Stoles) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the international Bureau later (j^/y<fc;_ * __rα..ι_iiιre_ _fc.fi--- uια-tf (.;, 'Accession Number of Deposit') For International Bureau use oniy I j This sheet was received by the International Bureau on: Authoπzed officer Applicant's or agent's file .201 International applications r /T) K 9 4 / 0 0 2 7 3 reference number INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule I3bis) A. The indications made below relate to the microorganism referred to in the description 7 ,. 28-31 on page , line __________________ B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet X Name of depositary institution Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) Address of depositary institution (including postal code and country) Maschroder Weg lb D-38124 Braunschweig Federal Republic of Germany Date of deposit Accession Number 28 June 1993 DSM 8379 C. ADDITIONAL INDICATIONS (leave blank ifnol applicable) This information is continued on an additional sheet ] As regards the respective Patent Offices of the respective designated states, the applicant requests that a sample of the deposited microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if Vindications are not for all designated Stales) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the international Bureau later ispcάfyt egencτalnaιureoftltoι'ndicaύonse.g., 'Accession Number of Deposit') For International Bureau use only I I This sheet was received by the International Bureau on: Authorized officer Applicant's or agent's file 1201 International application . > -'CT /TW Q A / D fl 9 7 "- Ϊ reference number INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule I3bis) The indications made below relate to the microorganism referred to in the description 8 ,. 15-20 on page ____________________ , line ____________________ B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | | Name of depositary institution Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) Address of depositary institution (including postal code and country) Maschroder eg lb D-38124 Braunschweig Federal Republic of Germany- Date of deposit Accession Number 30 June 1993 DSM ACC2134 C. ADDITIONAL INDICATIONS (leave blank ifnol applicable) This information is continued on an additional sheet ~ As regards the respective Patent Offices of the respective designated states, the applicant requests that a sample of the deposited microorganisms only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if tlie indications are not for all designated Stales) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the international Bureau later (spcαfythegenminαtureofιteindιcαuonse.g v 'Accession Number of Deposit ") For International Bureau use only [ | This sheet was received by the international Bureau on: Authoπzed officer

Claims: CLAIMS 1. A vaccine for immunizing an animal, including a human being, against tuberculosis caused by mycobacteria belonging to the tuberculosis-complex, comprising as the effective component at least one at least partially purified polypeptide, which is released from metabolizing mycobacteria belonging to the tuberculosis complex and can be isolated from short-term filtrates from such mycobacteria grown as shaken cultures for 7 days, and has a molecular weight in the range from about 3 kDa to about 16 kDa or in the range from about 20 kDa to about 40 kDa as determined by analysis by SDS-PAGE and silver staining, and induces a release of IFN-γ from reactivated memory T-lymphocytes withdrawn from a C57B1/6J mouse within 4 days after the mouse has been rechallenge infected with mycobacteria belonging to the tuberculosis complex, the induction performed by the addition of the polypeptide to a suspension comprising about 200.000 reactivated memory T-cells per ml, the addition of the polypeptide resulting in a concentration of 1 μg polypeptide per ml suspension, the release of IFN-γ being assessable by determination of IFN-γ in supernatant harvested 2 days after the addition of the polypeptide to the suspension, or an analogue and/or subsequence of the polypeptide, said analogue and/or subsequence being immunologically equivalent to the polypeptide with respect to the ability of evoking a protective immune response against tuberculosis or with respect to the ability of eliciting a delayed type hypersen- sitivity reaction, said polypeptide optionally being coupled to a pharmaceuti¬ cally acceptable carrier or vehicle. 2. A vaccine according to claim 1, wherein the polypeptide has a molecular weight in the range of about 4 to about 14 kDa, such as in the range of about 6 to 10 kDa, and preferab¬ ly in the range of about 5 to 6 kDa, in the range of about 6 to 7 kDa, or in the range of about 7 to 8 kDa. 3. A vaccine according to claim 1, wherein the polypeptide has a molecular weight in the range of about 22 to about 38 kDa, such as in the range of about 20 to about 32 kDa or in the range of about 35 to about 40 kDa; in the range of about 22 kDa to about 30 kDa or in the range of about 36 to about 39 kDa; in the range of about 24 kDa to about 28 kDa or in the range of about 36 to about 38 kDa; or in the range of about 25 kDa to about 27 kDa or in the range of about 37 to about 38 kDa. 4. A vaccine according any of the preceding claims, wherein the polypeptide is produced by the lysogenic E. coli strain designated AA227 which has been deposited 28 June 1993 with the collection of Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) under the accession number DSM 8378 in accordance with the provisions of the Budapest Treaty, - is produced by the lysogenic E. coli strain designated AA242 which has been deposited 28 June 1993 with the collection of Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) under the accession number DSM 8379 in accordance with the provisions of the Budapest Treaty, or reacts in a western blot assay with a monoclonal anti¬ body, HYB76-8, said antibody being produced by the hybridoma cell line designated HYB 76-8 0,5/br C8 0,25/br B3 which has been deposited 30 June 1993 with the collection of Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) under the accession number DSM ACC2134 in accordance with the provisions of the Budapest Treaty. 5. A vaccine according to any of the preceing claims, wherein the polypeptide comprises an amino acid sequence homologous to the amino acid sequence shown in SEQ ID NO: 2 or homologous to an analogue and/or subsequence of SEQ ID NO: 2. 6. A vaccine according to claim 5, wherein the degree of homology is at least 80%, such as at the least 90%. 7. A vaccine according to any of the preceding claims, where¬ in the polypeptide has been encoded by a nucleic acid frag- ment, which fragment 1) hybridizes with a DNA fragment comprising the nucleotide sequence shown in SEQ ID NO: 1 or a specific part thereof or with a DNA fragment complementary thereto, preferably under stringent hybridization conditions and/or 2) encodes a polypeptide, the amino acid sequence of which is at least 80% homologous with the amino acid sequence shown in SEQ ID NO: 2, and/or 3) constitutes an effective subsequence of said DNA sequence. 8. A vaccine according to any of the preceding claims, where¬ in the polypeptide or the analogue and/or subsequence thereof is capable of evoking a substantial and specific acquired immune resistance in a mouse or guinea pig against tuberculo¬ sis caused by mycobacteria belonging to the tuberculosis complex, which acquired immune resistance corresponds to at least 20% of the protective immune resistance elicited by Mycobacterium bovis BCG, as assessed by the observed reduc¬ tion in mycobacterial counts from spleen, lung or other organ homogenates isolated from the mouse or guinea pig receiving a challenge infection with a virulent strain of M. tuberculo- sis. 9. A vaccine according to claim 8 wherein the acquired immune resistance corresponds to at least 50% of the protective immune response elicited by M. bovis BCG, such as at least 60%, at least 80%, at least 90%, at least 100%, or at least 110%. 10. A vaccine according to any of the preceding claims, further comprising an adjuvant substance, such as DDA, Quil A, poly I:C, Freund's incomplete adjuvans, IFN-γ, IL-2, and IL-12. 11. A vaccine according to any of the preceding claims, wherein the IFN-γ released from the memory T-lymphocytes is at least 1500 pg/ml, such as 2000 pg/ml, preferably 3000 pg/ml. 12. A vaccine according to any of the preceding claims, further comprising any other polypeptide derived from a bacterium belonging to the tuberculosis complex, such as the ST-3 reactive polypeptide, the PV-2 reactive polypeptide, MPB64, MPT64, and MPB59. 13. A vaccine according to any of the preceding claims, comprising at least two different polypeptides, each polypeptide being as defined in claims 1-9. 14. A vaccine according to claim 13, comprising 3-20 diffe¬ rent polypeptides, each polypeptide being as defined in claims 1-9. 15. A vaccine for immunizing an animal, including a human being, against tuberculosis caused by mycobacteria belonging to the tuberculosis complex, comprising as the effective component a microorganism, wherein at least one copy of a DNA fragment comprising a DNA sequence encoding a polypeptide as defined in any of claims 1-9 or an analogue and/or subsequence thereof has been incorporated into the genome of the microorganism in a manner allowing the microorganism to express and optionally secrete the polypeptide. 16. A vaccine according to claim 15, wherein the microorga¬ nism is a bacterium. 17. A vaccine according to claim 16, wherein the bacterium is selected from the group consisting of the genera Mycobacteri ¬ um, Salmonella, Pseudomonas and Eschericia. 18. A vaccine according to claim 17, wherein the microorga¬ nism is Mycobacterium bovis BCG, such as Mycobacterium bovis BCG strain: Danish 1331. 19. A vaccine according to any of claims 15-18, wherein at least 2 copies of a DNA fragment encoding a polypeptide as defined in claims 1-9 are incorporated into the genome of the microorganism. 20. A vaccine according to claim 19 wherein the number of copies is at least 5. 21. A vaccine according to any of claims 15-20, wherein the DNA fragment which has been incorporated in the microorganism 1) hybridizes with a DNA fragment comprising the DNA sequence shown in SEQ ID NO: 1 or a specific part thereof or with a DNA fragment complementary thereto, preferably under stringent hybridization conditions and/or 2) encodes a polypeptide, the amino acid sequence of which is at least 80% homologous with the amino acid sequence shown in SEQ ID NO: 2, and/or 3) constitutes an effective subsequence of said DNA sequence. 22. A vaccine according to claim 21, wherein the DNA-sequence encodes a polypeptide, the amino acid sequence of which is at least 90% homologous with the amino acid sequence shown in SEQ ID NO: 2 or with a subsequence thereof. 23. A vaccine according to claim 22 wherein the DNA-sequence encodes the polypeptide with the amino acid sequence shown in SEQ ID NO: 2, or encodes a subsequence thereof. 24. A substantially pure polypeptide as defined in any of claims 1-9 or encoded by a DNA fragment as defined in any of claims 20-23 or an immunologically equivalent analogue and/or subsequence thereof. 25. A polypeptide according to claim 24, which comprises a T- cell epitope, such as an epitope for a T-helper cell. 26. A nucleic acid fragment as defined in any of claims 20-23 or encoding a polypeptide as defined in claim 24 or 25. 27. A nucleic acid fragment according to claim 26, which is a DNA-fragme t. 28. A nucleic acid fragment according to claim 27, which comprises the DNA sequence of SEQ ID NO: 1 or an analogue and/or subsequence thereof. 29. A composition for diagnosing tuberculosis, comprising a polypeptide as defined in claim 24 or 25, or a nucleic acid fragment as defined in any of claims 26-28, optionally in combination with a means for detection. 30. A method for diagnosing tuberculosis, comprising the use of a polypeptide according to claim 25 or 25 or a nucleic acid fragment as defined in any of claims 26-28. 31. A method of diagnosing tuberculosis caused by Mycobacte¬ rium tuberculosis, Mycobacterium africanum or Mycobacterium bovis in an animal, including a human being, comprising intradermally injecting, in the animal, a pharmaceutical composition containing a polypeptide according to claim 24 or 25, a positive skin response at the location of injection being indicative of the animal having tuberculosis, and a negative skin response at the location of injection being indicative of the animal not having tuberculosis. 32. A method for immunising an animal, including a human being, against tuberculosis caused by mycobacteria belonging to the tuberculosis complex, comprising administering the vaccine according to any of claims 1-23 to the animal. 33. A method according to claim 32, wherein the vaccine is administered intravenously, intraperitoneally, intracutaneously or intramuscularly. 34. A monoclonal antibody, which is substantially specifi¬ cally reacting with a polypeptide according to claim 24 or 25 in an immuno assay, or a specific binding fragment of said antibody. 35. An monoclonal antibody according to claim 34, which is expressed by the hybridoma cell line designated HYB 76-8 0,5/br C8 0,25/br B3 which has ' been deposited 29 June 1993 with the collection of Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) under the accession number DSM ACC2134 in accordance with the provisions of the Budapest Treaty or a specifically binding fragment of said antibody. 36. A nucleic acid fragment encoding the antibody according to claim 34 or 35. 37. A nucleic acid fragment according to claim 36, which is contained in the deposited hybridoma cell-line according to claim 4. 38. A replicable vector which expresses a polypeptide accor¬ ding to claim 24 or 25. 39. A vector according to claim 38, which is selected from the group consisting of vira, bacteriophages, plasmids, cosmids and microchromosomes. 40. A transformed cell harbouring at least one vector as defined in claim 38 or 39. 41. A transformed cell according to claim 40, which is a bacterium belonging to the tuberculosis complex. 42. A transformed cell according to claim 41, which is a M. tuberculosis bovis BCG cell. 43. A cell which is selected from the group consisting of the lysogenic E. coli strains AA227 and AA242 which have been deposited 28 June 1993 with the collection of Deutsche Sam- mlung von Mikroorganismen und Zellkulturen GmbH (DSM) under the accession numbers DSM 8378 and DSM 8379, respectively, in accordance with the provisions of the Budapest Treaty. 44. A cell according to any of claims 40-43, which expresses a polypeptide according to claim 24 or 25. 45. A method for producing a polypeptide according to claim 24 or 25, comprising inserting a DNA fragment as defined in claim 26 into a vector which is able to replicate in a host cell, introducing the resulting recombinant vector into the host cell, culturing the host cell in an appropriate culture medium under appro¬ priate conditions for expressing the polypeptide, and recove¬ ring the polypeptide from the host cell or culture medium, or isolating the polypeptide from a short-term culture filtrate as defined in claim 1. 46. A method for producing a vaccine according to any of claims 1-23 or 46, comprising preparing or isolating a polypeptide as defined in claim 24 or 25, and solubilizing or dispersing the polypeptide in a medium for a vaccine, and optionally adding other M. tuberculosis antigens and/or an adjuvant substance, or cultivating a cell according to any of claims 40-42, and transferring the cells to a medium for a vaccine, and optionally adding an adjuvant substance. 47. A vaccine comprising a nucleic acid fragment according to any of claim 26-28, the vaccine effecting in vivo expression of antigens by an animal, including a human being, to whom the vaccine has been administered, the amount of expressed antigens being effective to confer substantially increased resistance to infections with mycobacteria of the tuberculo¬ sis complex in an animal, including a human being. 48. A method of immunizing an animal, including a human being, against tuberculosis, comprising administering the vaccine according to claim 47 to the animal.

Inventors: ANDERSEN AASE BENGAARD, ANDERSEN PETER, HASLOEV KAARE, SOERENSEN ANNE LUND, ANDERSEN, AASE, BENGAARD, ANDERSEN, PETER, HASLOEV, KAARE, SOERENSEN, ANNE, LUND
Applicants: ANDERSEN AASE BENGAARD, ANDERSEN PETER, HASLOEV KAARE, SOERENSEN ANNE LUND, STATENS SERUMSINSTITUT, ANDERSEN, AASE, BENGAARD, ANDERSEN, PETER, HASLOEV, KAARE, SOERENSEN, ANNE, LUND
Application year: 1994
Main classification: C12N15
IPC codes: A61K39, C07K14